Biology subjects

Rejzek, M.

Publications and source records attributed to Rejzek, M..

9 recordsLinked to original sources

bioRxiv2026

Semisynthesis of Oxalyl-Coenzyme A for Enzymatic Assays

Oxalyl-coenzyme A (oxalyl-CoA) is a key intermediate in oxalate metabolism in plants, fungi and oxalate-degrading bacteria, but its limited availability has restricted biochemical investigations of oxalyl-CoA-dependent enzymes. Here, we describe a practical semisynthetic procedure for the preparation of oxalyl-CoA based on rapid oxalyl transfer from S-oxalyl p-thiocresol to coenzyme A. The reaction was monitored directly by 1H NMR spectroscopy, allowing optimisation of pD and reaction conditions. Following removal of thiocresol and purification by reversed-phase HPLC, oxalyl-CoA was obtained in 39% yield as determined by quantitative 1H NMR. The product was characterised by high-resolution electrospray mass spectrometry and comprehensive 1H, 13C and 31P NMR spectroscopy, confirming its structure unequivocally. During the study, the limited stability of oxalyl-CoA in aqueous solution was documented, leading to recommendations for its purification and storage. The semisynthetic protocol provides a convenient source of analytically pure oxalyl-CoA suitable for biochemical assays and supplies reference spectroscopic data for its unambiguous identification. The biological utility of the semisynthetic oxalyl-CoA was demonstrated by its application as an acyl donor substrate in assays of PnBAHD15, enabling quantitative kinetic characterisation of the enzyme and illustrating its suitability for biochemical studies of oxalyl-CoA-dependent enzymes.

biochemistry↗

bioRxiv2026

Repurposing a chromosome segregation ParB-CTPase fold into an ATPase toxin for contact-dependent growth inhibition in plant and animal pathogens

Bacterial competition drives the evolution of antibacterial mechanisms, yet how new activities arise remains poorly understood. A major route to innovation is the reuse of pre-existing genetic systems, whereby conserved protein modules are repurposed in new biological contexts to generate new capabilities. Here, we show that the ParB-CTPase fold, a conserved nucleotide-binding module best known for its role in chromosome segregation, can be functionally repurposed as an antibacterial toxin. We identify ToxB, a ParB-like domain embedded within the polymorphic toxin region of contact-dependent inhibition systems and show that it functions as a potent antibacterial effector. Structural and biochemical analyses reveal that ToxB retains the core architecture of the ParB-CTPase fold but lacks DNA-binding capability and preferentially binds ATP. This shift in nucleotide specificity underpins a distinct mode of action, in which ATP binding and hydrolysis trigger rapid nucleoid compaction, chromosome segregation defects, oxidative stress, cell chaining, and ultimately cell lysis. ToxB also exhibits toxic activity in plant cells, suggesting that it targets conserved cellular processes. Together, these findings provide direct experimental evidence that the ParB-NTPase fold is biologically versatile and can be repurposed for biological roles fundamentally distinct from its ancestral function in DNA segregation.

microbiology↗

bioRxiv2026

Efficient in vitro refactoring and biosynthetic gene cluster amplification for the overproduction and accelerated discovery of anticancer thioamitides

Thioamitides, a class of highly modified bacterial ribosomally synthesised and post-translationally modified peptides (RiPPs), have potent activities against multiple cancer cell lines. Among these compounds, the structurally divergent thioalbamide combines promising in vivo antiproliferative activity with a superior chemical stability respect to its counterparts. However, thioalbamide is produced in low yields by its genetically intractable native producer and its biosynthetic pathway was initially not productive when transferred into the heterologous host Streptomyces coelicolor M1146. These circumstances substantially hamper to increase the production of this promising compound. Here, we show how in vitro Gibson-like assemblies can be employed for the quick and efficient refactoring of the thioalbamide biosynthetic gene cluster (BGC), leading to substantially increased levels of production in S. coelicolor M1146 through a prioritised selection of promoters. Via this work, PtsrA and PgroEL2 were identified as beneficial additions to the Streptomyces synthetic biology toolbox. We then assessed bacterial genomes for biosynthetic gene clusters (BGCs) predicted to produce thioalbamide-like compounds with improved hydrophilicity. This rational discovery campaign led to the identification a silent thioamitide BGC encoding a thioalbamide-like core peptide but clustered with additional tailoring enzymes, including a previously unknown cupin-fold protein. Applying the refactoring strategy together with the simultaneous expression of multiple BGC copies, we characterised the product of this pathway, thiocupinamide, a polyhydroxylated thioamitide closely related to thioalbamide. We show that thiocupinamide has potent anticancer and antibacterial activities.

synthetic biology↗

bioRxiv2025

MtrAB activates ectoine production and triggers sporulation in response to osmotic stress in Streptomyces venezuelae

The MtrAB two-component system is a master regulator of antibiotic biosynthesis in Streptomyces species. MtrA is also required for sporulation under certain growth conditions, which means that on some growth media {Delta}mtrA mutant colonies do not produce aerial hyphae or spores. These mutants are referred to as (conditionally) bald because they lack the hairy appearance of wild-type colonies. Here we report that S. venezuelae NRRL B-65442 {Delta}mtrA is bald on R2YE agar but sporulates normally on MYM agar and we demonstrate that this is caused by the presence of 10.2% sucrose in R2YE. Consistent with this, we found that adding 10.2% sucrose to MYM agar also inhibits sporulation of the {Delta}mtrA mutant. Proteomics combined with DNA binding studies revealed that MtrA directly activates the expression of the key developmental regulator genes bldM and whiI on R2YE but not MYM agar. BldM and WhiI work together to activate genes required for aerial hyphae production and sporulation. Crucially, over-expression of bldM-whiI in the {Delta}mtrA mutant restored normal sporulation in the presence of 10.2% sucrose. We hypothesised that MtrAB must sense and respond to osmotic stress and consistent with this we found that MtrA directly activates biosynthesis of the compatible solute and osmoprotectant ectoine on growth media containing 10.2% sucrose. We propose a model in which high concentrations of sucrose induce osmotic stress in Streptomyces species and this activates MtrAB. The response regulator MtrA then directly activates expression of the ectABCD operon to switch on ectoine biosynthesis and expression of bldM and whiI to trigger entry into sporulation. Impact statementStreptomyces species have complex developmental life cycles that start with spore germination and outgrowth of an actively growing substrate mycelium. Environmental stresses including nutrient starvation and osmotic stress trigger the production of aerial hyphae that undergo cell division to form spores. These spores are more resistant to environmental stresses and can stay viable in the soil until conditions improve. Here we show that MtrAB is responsible for sensing and responding to osmotic stress and we demonstrate that it directly controls the biosynthesis of ectoine and the developmental transition to sporulation through direct activation of BldM and WhiI. This work provides new insight into how environmental cues control the development of Streptomyces bacteria. Data summaryThe S. venezuelae NRRL B-65442 genome sequence is available at NCBI (Reference Sequence NZ_CP018074.1) and can be viewed at http://strepDB.streptomyces.org.uk (select vnz chromosome from the drop-down list). The ChIP-seq data (GEO accession number CP018074) used to map MtrA binding sites on the S. venezuelae NRRL B-65442 chromosome and the differential RNA-seq data (accession number GSE81104) used to map global transcript start sites throughout the life cycle were generated for earlier studies (1,2). The tandem mass tag proteomics data are available via ProteomeXchange with identifier PXD069151. The ReDCaT SPR data is included in the manuscript and the supplementary information. Protocols are freely available at http://actinobase.org (3) and strains and plasmids are available from http://streptomyces.org.uk/strepstrains. The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

microbiology↗

bioRxiv2024

A Necrotizing Toxin Promotes Pseudomonas syringae Infection Across Evolutionarily Divergent Plant Lineages

The Pseudomonas syringae species complex harbors a diverse range of plant pathogenic bacteria. While much of the current understanding of P. syringae is centered on interactions with flowering plants, much less is known about infection in evolutionarily divergent non-flowering lineages. Here, we took a comparative evolutionary approach to understand how P. syringae infects distantly related plants. We identify broad host P. syringae isolates causing significant disease in the liverwort Marchantia polymorpha, the fern Ceratopteris richardii, and the flowering plant Nicotiana benthamiana, which last shared a common ancestor over 500 million years ago. We demonstrate that phytotoxin enriched isolates belonging to the phylogroup 2 clade of the P. syringae species complex are particularly virulent in non-flowering plants, relying on a combination of type-3 secreted effector proteins and the lipopeptide phytotoxin syringomycin. The application of purified syringomycin promotes necrosis in diverse host tissues and activates conserved genes associated with redox regulation and cell death. Toxin-deficient phylogroups normally unable to infect Marchantia thalli exhibit enhanced bacterial growth when supplemented with exogenous syringomycin, further highlighting its role as a host-range defining factor in Pseudomonas. Collectively our research reveals a key role for the lipopeptide syringomycin in promoting Pseudomonas colonization, which works in concert with type-3 effector proteins to antagonize an exceptionally wide spectrum of land plants.

plant biology↗

bioRxiv2021

Spinning sugars in antigen biosynthesis: a direct study of the Coxiella burnetii and Streptomyces griseus TDP-sugar epimerases

The sugars streptose and dihydrohydroxystreptose (DHHS) are unique to the bacteria Streptomyces griseus and Coxiella burnetii respectively. Streptose forms the central moiety of the antibiotic streptomycin, whilst DHHS is found in the O-antigen of the zoonotic pathogen C. burnetii. Biosynthesis of these sugars has been proposed to follow a similar path to that of TDP-rhamnose, catalysed by the enzymes RmlA/RmlB/RmlC/RmlD. Streptose and DHHS biosynthesis unusually require a ring contraction step that might be performed by the orthologues of RmlC or RmlD. Genome sequencing of S. griseus and C. burnetii proposed the StrM and CBU1838 proteins respectively as RmlC orthologues. Here, we demonstrate through both coupled and direct observation studies that both enzymes can perform the RmlC 3,5 double epimerisation activity; and that this activity supports TDP-rhamnose biosynthesis in vivo. We demonstrate that proton exchange is faster at the 3 position than the 5 position, in contrast to a previously studied orthologue. We solved the crystal structures of CBU1838 and StrM in complex with TDP and show that they form an active site highly similar to previously characterised enzymes. These results further support the hypothesis that streptose and DHHS are biosynthesised using the TDP pathway and are consistent with the ring contraction step being performed on a double epimerised substrate, most likely by the RmlD paralogue. This work will support the determination of the full pathways for streptose and DHHS biosynthesis.

biochemistry↗

bioRxiv2021

CTP regulates membrane-binding activity of the nucleoid occlusion protein Noc

ATP and GTP-dependent molecular switches are extensively used to control functions of proteins in a wide range of biological processes. However, CTP switches are rarely reported. Here, we report that a nucleoid occlusion protein Noc is a CTPase enzyme whose membrane-binding activity is directly regulated by a CTP switch. In Bacillus subtilis, Noc nucleates on 16-bp NBS sites before associating with neighboring non-specific DNA to form large membrane-associated nucleoprotein complexes to physically occlude assembly of the cell division machinery. By in vitro reconstitution, we show that (i) CTP is required for Noc to form the NBS-dependent nucleoprotein complex, and (ii) CTP binding, but not hydrolysis, switches Noc to a membrane-active state. Overall, we suggest that CTP couples membrane-binding activity of Noc to nucleoprotein complex formation to ensure productive recruitment of DNA to the bacterial cell membrane for nucleoid occlusion activity.

microbiology↗

bioRxiv2021

Chemical synthesis and purification of a non-hydrolyzable CTP analog CTPγS

Slowly- or non-hydrolyzable analogs of ATP and GTP, for example adenosine 5-({gamma}-thio)triphosphate (ATP{gamma}S) and guanosine 5-({gamma}-thio)triphosphate (GTP{gamma}S), have been widely employed to probe the roles of ATP and GTP in biosystems, and these compounds are readily available from commercial sources. In contrast, cytosine 5-({gamma}-thio)triphosphate (CTP{gamma}S) is not widely available commercially. The recent discovery of ParB as the founding member of a new class of CTPase enzyme (Osorio-Valeriano et al., 2019; Soh et al., 2019) and the possibility of multiple other undiscovered CTPases necessitate the use of the CTP{gamma}S to elucidate the roles of CTP hydrolysis in these systems. Here, we adapt a procedure for the synthesis of modified phosphoanhydrides (Hofer et al., 2015) to chemically synthesize and purify a milligram-scale of CTP{gamma}S.

biochemistry↗

bioRxiv2020

Dissecting the toxicity and mitigating the impact of harmful Prymnesium blooms in the UK waters of the Norfolk Broads

Prymnesium parvum is a toxin-producing microalga that causes harmful algal blooms (HABs) globally, frequently leading to massive fish kills that have adverse ecological and economic implications for natural waterways and aquaculture alike. The dramatic effects observed on fish are likely due to algae-produced polyether toxins, known as the prymnesins, but these compounds had not been detected in environmental samples, which has resulted in ambiguity about the true ichthyotoxic entities. Using qPCR, we found elevated levels of both P. parvum and its lytic virus, PpDNAV-BW1, in a fish-killing bloom on the Norfolk Broads, United Kingdom, in March 2015, a site historically plagued by P. parvum blooms. 16S rRNA gene sequencing confirmed that P. parvum dominated the bloom microbial community and that the microbial species diversity changed after recovery of the ecosystem. We also detected, for the first time, the recently discovered B-type prymnesin toxins in Broads waterway samples and gill tissue isolated from a dead fish taken from the study site. Furthermore, Norfolk Broads P. parvum isolates unambiguously produced B-type toxins in laboratory-grown cultures. In addition, a 2-year longitudinal study of the Broads study site showed P. parvum blooms positively correlated with increased temperature and that PpDNAV plays a significant role in P. parvum bloom demise in this natural setting. Finally, we used a field trial to show that treatment with low doses of hydrogen peroxide represents an effective strategy to mitigate blooms of P. parvum in enclosed water bodies.

microbiology↗